Table 1.
Sequences of primers and probes.
Table 2.
In-vitro cytokine stimulation.
Figure 1.
Composition of the synthetic standard gene comprising all target cytokines (IL-2, IL-4, IL-8, TNF-α, IFN-α, IL-6, IL-1β) and internal reference genes (β-Actin, GAPDH, HPRT (Hypoxanthin-Guanin-Phosphoribosyltransferase), starting with the T7-promotor sequence and concluding with the NOD1 restriction site as initial point for linearization and transformation to RNA.
Each target cytokine was included with a nucleotide overhang of approximately 50 base pairs (bp) prior to forward primer sequence. In total, the synthetic standard gene comprises 1464 bp.
Table 3.
Samples from different animal trials (n = 402) used for assay validation.
Figure 2.
Detection of normalized TNF-α gene expression (ΔΔCq) in leukocyte samples from CSFV infected pigs.
Samples were obtained from two different pig breeds at days 0, 2, 4, and 7 post infection (dpi). Results are given as mean values: in total from all animals (TNF-α overall mean) and separately for each breed (Mean Breed 1, Mean Breed 2). Bars indicate standard deviations.
Figure 3.
Detection of normalized IL-8 gene expression (ΔΔCq) in leukocyte samples from CSFV infected pigs.
Samples were obtained from two different pig breeds at days 0, 2, 4, and 7 post infection (dpi). Results are given as mean values: in total from all animals (IL-8 overall mean) and separately for each breed (Mean Breed 1, Mean Breed 2). Bars indicate standard deviations.
Figure 4.
Comparative amplifications of 10-fold diluted standard RNA ranging from 2×102 to 2×107 copies/µl of single target and multiplex assays of all target cytokines (FAM).
The vertical axis demonstrate fluorescence levels (relative fluorescence units, RFU), the horizontal axis shows the number of cycles. Comparative illustrations of end fluorescence levels of standard curves are shown illustrating higher end fluorescences in single target compared triplex assays in which target cytokines were detected simultaneously with β-Actin (HEX) and GAPDH (TR).